Recent studies indicate that respiratory viruses, including Respiratory Syncytial Virus (RSV), disrupt epithelial barrier structure and function, but the mechanisms involved and consequences for airway inflammation remains poorly understood. The long-term goal of our studies is to better understand how airway epithelial cell barrier structure and function are affected by viral infection and how modifying the involved mechanisms restore barrier integrity and dampen inflammatory responses. Our recent publication revealed that, in cell culture, RSV infection caused disruption of tight junction structure and function. Our objective in this particular application is to investigate the molecula mechanism and signaling pathways involved in RSV-induced barrier disruption. The strength of our approach is that we utilize four highly complementary models including a well-characterized and manipulable human bronchial epithelial cell line, primary human and mouse airway epithelial cells, and cutting-edge mouse models; each serves to examine different key aspects of proposed mechanisms. The central hypothesis is that RSV infection prompts airway epithelial barrier dysfunction via endocytosis of apical junctional complexes (AJC) in a Protein Kinase D (PKD)-dependent manner. Guided by strong preliminary data, this hypothesis will be tested by pursuing two specific aims: 1) Test the hypothesis that PKD- dependent endocytosis is essential for RSV-induced airway epithelial barrier dysfunction; 2) Test the hypothesis that RSV causes sustained leaky airway in a PKD-dependent manner. In Aim 1, we will elucidate endocytic pathways, cytoskeleton remodeling, and PKD activation triggered by RSV. In Aim 2, we will use a well-characterized mouse model of wild type, and cutting-edge conditional deletion of PKD to investigate association of junction dysfunction, lung inflammation, and viral replication. Our proposal is innovative because it is the first comprehensive analysis of role of RSV infection and PKD pathway in the lung. Furthermore, it will provide an innovative assay of outside/in airway permeability by using inhaled mannitol, which has strong translational potential. The proposed research is significant, because it will provide essential knowledge about a clinically relevant virus with poorly understood pathogenesis. The overall K08 application is designed to build a foundation of technical, intellectual and leadership skills required to transition into independence. The career development plan will help me strengthen my knowledge in virology, and barrier immunology, develop expertise in studying cell signaling, and endocytosis, acquire essential skills in utilizing genetically modified mice, and define my niche as an independent physician scientist in the field of respiratory biology. My mentors and advisory committee will monitor adherence to the detailed productivity and developmental progress timeline. My commitments to a research career, strong mentorship, unique dedication of Pediatrics Department in training the next generation of physician-scientists, and outstanding collaborative environment for respiratory virus research at the University of Rochester will provide the necessary tools with which I can build my career.